The suppressor of cytokine signaling (SOCS)- genes, SOCS1 and SOCS3, are critical targets of signal transducer and activator of transcription (STAT) pathways, providing normal negative feed-back control of signaling by cytokines and growth factors. Consistent with STAT activation in many tumor cells, SOCS1 expression is silenced in human cancer and enforced SOCS1 expression inhibits tumorigenesis. SOCS3 is closely related to SOCS1, but its regulation and role in oncogenesis remain largely unknown. Our preliminary studies find that cells transformed by Lck exhibit active STATS but fail to express both SOCS genes. In contrast to SOCS1 silencing by DNA methylation, defect of SOCS3 expression may be linked to the absence of STAT5b phosphorylation on non-conserved serine sites identified by our mass spectrometry. Additional preliminary studies show that SOCS3 associates with Lck in cells and becomes tyrosine-phosphorylated, and ectopic SOCS3 expression results in short-term growth suppression of Lck-transformed cells. Our long-term objectives are to determine how expression of different SOCS genes is disrupted in malignancies and to gain new mechanistic insights of their roles as tumor suppressors in various tumor models. This proposal focuses on the regulation of SOCS3 gene expression and protein functions. Aim 1 will determine the role of novel STAT5b serine phosphorylation in SOCS3 expression. We propose to define the functional STAT5b serine phosphorylation sites and elucidate its role in STAT5b-dependent assembly of transcriptional complex on the SOCS3 promoter in the context of chromatin. Aim 2 will test our hypothesis that SOCS3 and Lck proteins mutually regulate each other's functions and characterize the underlying mechanism. We further propose that a tyrosine mutant of SOCS3 is more potent than wild-type SOCS3 in suppressing Lck-mediated oncogenesis. In Aim 3, different SOCS3 proteins will be expressed in Lck-transformed LSTRA cells to evaluate their tumor-suppressing activities both in culture and in mice. The possibility to restore endogenous SOCS3 expression will also be tested with potential gain-of-function STAT5b mutants in LSTRA. Using Lck as a model system, we will start to define molecular details underlying the differences between SOCS1 and SOCS3 in their gene regulation and protein functions. Functional characterization of novel serine phosphorylation sites in STAT5b will further establish a new paradigm for its role in regulating STAT signaling and STAT-mediated gene expression. Relevance: SOCS proteins are normally produced by cells to turn off signals that stimulate their growth. Many cancer cells lose expression of these potential tumor suppressors. Studies proposed here will help us better understand why SOCS genes are not expressed in human cancer and how SOCS proteins may function differently in tumor cells. Collectively they will reveal potentially new targets for cancer therapy.